Vol. 334 No.

18 MOLECULAR MEDICINE 1185

MOLECULAR MEDICINE

ADENOVIRUSES AS GENE-DELIVERY
VEHICLES

J

AMES

M. W

ILSON

, M.D., P

H

.D.

F

OR gene therapy to realize its clinical potential,
there must be efcient and safe strategies of deliv-
ering therapeutic genes to somatic cells in vivo. Perhaps
this problem simply represents a special case of drug
delivery in which the therapeutic gene constitutes the
drug. But the relevance of traditional drug development
to technological developments in gene therapy is ques-
tionable. Recombinant genes that can independently ex-
press a therapeutic RNA or protein are extraordinar-
ily large and polar molecules 10,000 times larger
than traditional pharmaceutical agents. Moreover, the
administration of genes as therapy requires vehicles
so-called vectors that encapsulate the gene and guide
it to the target cell. The binding of the gene to the cell,
its internalization, the transport of the administered ge-
nome to the nucleus, and the expression of the gene all
constitute potential limitations of this process.
Recombinant viruses have generally been highly ef-
cient vectors. Murine retroviruses have been used ex-
tensively as vectors in preclinical and clinical models,
but their use has been restricted to ex vivo studies be-
cause of difculties in purifying and concentrating them
and the requirement that the target cells must divide in
order for retroviruses to infect them. In these systems,
autologous cells are modied genetically during culti-
vation in vitro. Vectors based on human adenoviruses
have shown more promise than retroviruses for in vivo
gene delivery because they can transfer recombinant
genes efciently into a wide variety of dividing and non-
dividing cells.
Human adenoviruses were initially evaluated as vec-
tors to treat cystic brosis, because of their tropism for
pulmonary epithelial cells. The more than 40 serotypes
of adenoviruses cause clinical syndromes ranging from
diarrhea to pharyngitis, none of which are usually se-
vere or associated with cancer. These nonenveloped vi-
ruses contain a 36-kb genome that consists of a series
of early genes, encoding regulatory proteins, and late
genes, which encode structural proteins. The adenovi-
rus is particularly attractive as a vector because it can
produce large amounts of highly puried recombinant
virus, which efciently infects differentiated, nondivid-
ing cells. Engineering the virus to make it a truly defec-
tive vector would require replacing the part of the viral
genome that encodes structural proteins with the ther-
apeutic gene. Difculties in producing a virus altered so
substantially instigated another strategy, one in which
the early adenovirus genes that activate other viral
genes are deleted (Fig. 1). In the absence of these reg-
ulatory genes, the virus should not replicate in vivo and
the late viral genes should remain dormant. These rst-
generation adenoviral vectors can propagate in vitro
and convey recombinant genes to many kinds of cells.
There has been extensive experience with such rst-
generation recombinant adenoviruses in cystic brosis.
In this autosomal recessive disorder a mutation disables
the cystic brosis transmembrane conductance regu-
lator (

CFTR

) gene. Defects in ion transport across pul-
monary epithelia follow, and they lead to recurrent
infection, bronchiectasis, and respiratory failure. Instil-
lation of adenoviral vectors containing the

CFTR

gene
into the airways of rodents culminates in extraordinar-
ily high expression of the recombinant gene in most ep-
ithelial cells of the conducting airways. These observa-
tions, as well as many studies of safety in nonhuman
primates, prompted phase 1 clinical trials of safety and
biologic efcacy (i.e., the efciency and stability of re-
combinant-gene expression) in patients with cystic -
brosis. This preliminary experience with adenoviral
vectors has revealed several themes. Initial concern
about the hazards of viral recombination, replication,
and shedding has not been realized. Inammation does

From the Institute for Human Gene Therapy and the Department of Molecular
and Cellular Engineering, University of Pennsylvania, and the Wistar Institute
both in Philadelphia. Address reprint requests to Dr. Wilson at the Institute for
Human Gene Therapy, 204 Wistar Institute, 3601 Spruce St., Philadelphia, PA
19104-4268.

1996, Massachusetts Medical Society.

Figure 1. Development of an Adenovirus as a Vector.
The genome of a replication-competent adenovirus is shown in
the nucleus of the upper epithelial cell. When the viral genome
enters the cell, the immediate early genes (

E1

) are expressed.
They activate the other important regulatory early genes,

E2,
E3,

and

E4.

In the second phase of replication, the late genes


L1

through

L5

are expressed, leading to the production of
more viruses and the death of the cell. In the lower cell, the ad-
enoviral genome has been developed as a vector. Deletion of
the

E1

genes renders the vector unable to replicate. In their
place the cystic brosis transmembrane conductance regulator
E2E4
Replication-
competent
virus
E1 L1L5
E2E4 L1L5
CFTR
Replication-
deficient
virus
CFTR
channel
Epithelial
cell

gene (

CFTR

), which encodes the CFTR channel, has been
inserted.


1186 THE NEW ENGLAND JOURNAL OF MEDICINE May 2, 1996

occur with rst-generation vectors in a dose-dependent
manner at the site of gene transfer, but there are differ-
ences among species in the quality and quantity of the
inammation. A consistent nding is that the expres-
sion of the therapeutic

CFTR

gene is only transient.
Moreover, attempts to restore expression by the admin-
istration of another dose of the vector are usually not
successful. Indeed, the efciency with which adenoviral
vectors transfer genes into airway epithelium has come
into question. These limitations have tempered the ini-
tial enthusiasm for rst-generation adenoviral vectors
in the treatment of cystic brosis.
Transient expression of the therapeutic gene is a sub-
stantial limitation of the rst-generation adenoviral vec-
tors in the genetic treatment of chronic diseases that
may require prolonged genetic correction. The pro-
tein product of the gene usually becomes undetectable
within two to three weeks, and gene expression after a
second administration of vector is inefcient or impos-
sible. These problems stem in part from the humoral
and cellular immune responses to the vector and vector-
infected host cells. These events resemble the immune
responses to any viral infection. The adenoviral pro-
teins or (in the case of patients with genetic defects) the
therapeutic gene product itself elicits cytotoxic T cells
that destroy the vector-infected cells (Fig. 2). The result
is loss of expression of the therapeutic gene and inam-
mation. In addition, antiviral antibodies develop that
can neutralize the vector when it is administered a sec-
ond time. To overcome these immunologic problems,
vectors have been engineered to minimize the expres-
sion of viral antigens. Another approach is to prevent
activation of T helper cells, an event that occurs pri-
marily at the time of vector delivery, by administering
an immunosuppressive drug along with the vector. Com-
bining an improved vector with transient immune mod-
ulation may sufce to overcome the problematic host
responses.
A controversy that has emerged from the initial clin-
ical trials of adenoviral vectors in cystic brosis con-
cerns the efciency of gene transfer in the airway. Clin-
ical studies of the instillation of vector into the lung
through a bronchoscope have demonstrated gene trans-
fer in airway cells when a low dose of vector is used, as
was found in animal models. Another type of delivery
restricted the vector to nasal epithelia. In this case the

Figure 2. Immune Responses to Adenovirus Vectors.
The vector enters a macrophage, and its genome takes up residence in the nucleus. The genome of the vector expresses viral pro-
teins that are presented by MHC class I molecules to CD8

T cells. The CD4



T



cells, activated by viral-capsid antigens, stimulate
the cytotoxic T lymphocytes, which destroy the genetically corrected target cell and provoke inammation. At the same time, B cells
are activated to secrete antibodies that neutralize the vector. These neutralizing antibodies prevent further administration of vector
from being successful.
B cell
CD4
T helper
cell
Cytotoxic
CD8 T cell
MHC
class II
Macrophage
Neutralizing
antibodies
Gene transfer blocked
Natural killer cell
Elimination
of cell
Vector
MHC
class I
Macrophage
Genome

Vol. 334 No. 18 MOLECULAR MEDICINE 1187

premise was that the nasal mucosa are relevant to the
epithelia of the intrapulmonary conducting airway and
are easily accessible for experimental manipulation.
Three independent groups of investigators found that
gene transfer with adenoviral vectors is extremely inef-
cient in unperturbed nasal mucosa affected by cystic
brosis. Studies of the biology of adenovirus entry into
cells have suggested an explanation for the difference
between nasal and pulmonary epithelia in the efciency
of gene transfer. Of the cellular receptors needed for
the entry of adenovirus vectors, one receptor is not
present in nasal mucosa but does occur in distal con-
ducting airways.
The ability of adenoviral vectors to deliver genes ef-
ciently to a wide variety of cells, dividing and quies-
cent, has been exploited in the development of gene
therapies for cancer. Features of rst-generation vectors
that limit their application in genetic diseases may ac-
tually prove benecial in the case of tumor vaccines de-
signed to deliver genes to tumor cells. For example, the
delivery of a gene for an immune regulatory cytokine
that amplies a specic antitumor cellular immune re-
sponse in vivo could nd clinical use. In another ap-
proach, the adenoviral vector delivers a gene that sen-
sitizes the tumor to a chemotherapeutic drug. Clinical
trials have begun for the treatment of mesothelioma
that are based on adenoviral-vectormediated gene
transfer of the thymidine kinase gene of the herpes sim-
plex virus, which converts the drug ganciclovir to a tox-
ic phosphorylated metabolite. This toxic effect is lim-
ited to cells undergoing mitosis, but it is extended
to nongenetically modied but contiguous tumor cells
through a mechanism called the bystander effect. The
limitations of rst-generation vectors, such as transient
expression of the therapeutic gene, are less problematic
in this kind of application.
The early experience with adenovirus vectors has de-
ned basic principles of in vivo gene therapy that will be
of generic importance to many vector systems. Immune
responses of the patient to the vector and the vector-
infected cell may preclude stable therapeutic-gene ex-
pression and vitiate the repeated dosing that the treat-
ment of chronic diseases may require. Revisions in the
vector to make it more stealth-like, combined with
transient immune suppression, will probably overcome
these problems. The use of adenoviral vectors for vac-
cines, such as in the treatment of certain types of can-
cers, may in fact be possible with existing techniques.
Broader applications of adenoviral vectors in gene ther-
apy will require modications that improve efciency
and promote targeting at the level of vector uptake, re-
combinant-gene expression, or both. There is every rea-
son to expect that the promising data generated with
adenoviral vectors in animal models will predict their
clinical uses. However, careful and objective trials in hu-
mans are needed to validate these hypotheses.

R

ECOMMENDED

R

EADING

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